Tanaka M, Katashima R, Murakami D, Adzuma K, Takahashi Y, Tomonari A, Iwahana H, Yoshimoto K, Itakura M
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Diabetologia. 1995 Apr;38(4):381-6. doi: 10.1007/BF00410274.
To understand the molecular basis of glucose concentration-responsive insulin synthesis and secretion from pancreatic islet beta cells, a group of pancreatic islet beta-cell-related cDNAs was cloned. A pair of cDNA libraries was constructed from a mouse pancreatic islet beta-cell line of MIN6, which was cultured in either high glucose or low glucose media. By applying a random cDNA sequencing approach, 503 and 395 independent species were obtained from a total of 1,011 and 762 clones in the high glucose and low glucose library, respectively. The unknown genes comprised the majority of about 70% independent clones in both libraries. In Northern blot analysis, 311 (69.4%) of 448 independent clones showed positive signals within 72 h of autoradiographic exposure. Surprisingly, 150 (48.2%) out of 311 positive clones showed positive signals to MIN6 cells, but not to NIH/3T3 fibroblasts. The expression level of three unknown clones were glucose-concentration dependent. Combination of a random cDNA sequencing approach and Northern blot analysis is useful to obtain a large number of novel genes and islet beta-cell-related genes.
为了解胰腺胰岛β细胞中葡萄糖浓度响应性胰岛素合成与分泌的分子基础，克隆了一组与胰腺胰岛β细胞相关的cDNA。从在高糖或低糖培养基中培养的小鼠胰腺胰岛β细胞系MIN6构建了一对cDNA文库。通过应用随机cDNA测序方法，在高糖文库和低糖文库中分别从总共1011个和762个克隆中获得了503个和395个独立的物种。未知基因在两个文库中约占独立克隆的70%。在Northern印迹分析中，448个独立克隆中的311个（69.4%）在放射自显影片曝光72小时内显示出阳性信号。令人惊讶的是，311个阳性克隆中的150个（48.2%）对MIN6细胞显示阳性信号，但对NIH/3T3成纤维细胞不显示阳性信号。三个未知克隆的表达水平取决于葡萄糖浓度。随机cDNA测序方法与Northern印迹分析相结合，对于获得大量新基因和与胰岛β细胞相关的基因很有用。
